Chan, Chi Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 103-127). / Abstracts in English and Chinese. / Thesis Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / 摘要 --- p.iv / Abstract --- p.vi / List of Abbreviations --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiii / Table of Content --- p.xiv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Classification of the aldehyde dehydrogenase superfamily --- p.1 / Chapter 1.2 --- Structures and catalytic mechanism of ALDH --- p.4 / Chapter 1.3 --- Multiple functions of ALDH --- p.8 / Chapter 1.4 --- Antiquitin - background and recent discoveries --- p.12 / Chapter 1.5 --- Aim of study --- p.19 / Chapter Chapter 2 --- Mitochondrial and Cytosolic Localizations of ALDH7A1 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Cell culture --- p.26 / Chapter 2.2.2 --- Subcellular fractionation --- p.26 / Chapter 2.2.3 --- Western blot analysis --- p.27 / Chapter 2.2.4 --- Flow cytometric analysis of mitochondria in WRL68 cells --- p.28 / Chapter 2.2.5 --- Transient transfection of various EGFP constructs --- p.29 / Chapter 2.2.6 --- Immunofluorescence staining --- p.31 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Presence of ALDH7A1 in cytosol and mitochondria in WRL68 cells --- p.33 / Chapter 2.3.2 --- Mitochondrial-targeting N-terminal sequence in ALDH7A1 --- p.34 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- In silico and in vitro subcellular localization studies on ALDH7A1 --- p.40 / Chapter 2.4.2 --- Significance of mitochondrial and cytosolic localizations of ALDH7A1 --- p.45 / Chapter 2.4.3 --- Comparison of animal ALDH7A and plant ALDH7B enzymes --- p.48 / Chapter Chapter 3 --- "ALDH7A1: A Potential Regulator for Cell Growth, Cell Cycle and a Potential Biomarker for Cancer (Stem) Cells" / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.55 / Chapter 3.2.1 --- Cell synchronization --- p.55 / Chapter 3.2.2 --- Semi-quantitative determination of DNA amount in synchronized cells --- p.55 / Chapter 3.2.3 --- Total protein extraction --- p.55 / Chapter 3.2.4 --- Western blot analysis --- p.57 / Chapter 3.2.5 --- Immunofluorescence staining --- p.57 / Chapter 3.2.6 --- Expression and purification of ALDH7A1 and its mutant --- p.57 / Chapter 3.2.7 --- Kinetic analysis of ALDH7A1 and its mutant --- p.58 / Chapter 3.2.8 --- Generation of native ALDH7 A1 and mutant for transfection --- p.58 / Chapter 3.2.9 --- Generation of stable cell line transfectants --- p.59 / Chapter 3.2.10 --- 2D cell culture and ultra-low attachment cell culture --- p.59 / Chapter 3.2.11 --- Collection of total cell lysates --- p.60 / Chapter 3.2.12 --- Western blot analysis --- p.60 / Chapter 3.2.13 --- Growth analysis --- p.61 / Chapter 3.2.14 --- Aldefluor assay --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Expression level of ALDH7A1 at different phases of the cell cycle --- p.62 / Chapter 3.3.2 --- Subcellular distribution of ALDH7A1 in synchronized cells --- p.64 / Chapter 3.3.3 --- Changes in the expression level of key cell cycle regulators and the growth rate after ALDH7A1 knockdown --- p.68 / Chapter 3.3.4 --- Absence of catalytic activity in the purified ALDH7A1 mutant C302S --- p.68 / Chapter 3.3.5 --- Over-expression of ALDH7A1 variants in HEK293 cells --- p.73 / Chapter 3.3.6 --- Growth rates of cells overexpressing different ALDH7A1 variants --- p.73 / Chapter 3.3.7 --- Expression level of ALDH7A1 in various 2D cell types and stem-like cells --- p.76 / Chapter 3.3.8 --- Aldefluor assay on cells over-expressing different ALDH7A1 variants --- p.79 / Chapter 3.4 --- Discussion --- p.82 / Chapter 3.4.1 --- Nuclear localization of ALDH7A1 --- p.82 / Chapter 3.4.2 --- Potential role of ALDH7A1 in cell cycle --- p.86 / Chapter 3.4.3 --- Non-catalytic role of ALDH in cell growth and development --- p.86 / Chapter 3.4.4 --- Relationship between ultra-low attachment culture and stem-like cells --- p.89 / Chapter 3.4.5 --- Up-regulation of ALDHs in cancer and CSCs and the evaluation of applicability of Aldefluor assay in CSC isolation --- p.93 / Chapter 3.4.6 --- Comparison on ALDH7A1 expression level in primary and stem-like cells --- p.98 / Chapter Chapter 4 --- Future Prospects / References --- p.103

Wong Wei-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 221-242). / Abstracts in English and Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract in Chinese --- p.iv / Abstract in English --- p.vi / List of abbreviations --- p.viii / List of figures --- p.xi / List of tables --- p.xv / Content: --- p.xvi / General introduction --- p.1 / Aldehyde dehydrogenase superfamily --- p.3 / Background of antiquitin --- p.5 / Plant antiqutins (ALDH7B) --- p.5 / Animal antiquitins (ALDH7A) --- p.8 / Human antiquitin information on NCBI --- p.14 / Rationale of studying the inducibility of annquitin and overexpression of it in HEK293 and HepG2 cells --- p.16 / Flowchart 1 Procedure of antiquitin expression studies in the HEK293 and HepG2 cells under stress --- p.19 / Flowchart 2 Procedure to study antiquitin expression in the HEK293 and HepG2 cells after in silico promoter search --- p.20 / Flowchart 3 Procedure to study antiquitin overexpressed HEK293 and HepG2 cells --- p.21 / Chapter Chapter 1 --- Inducibility of antiquitin in the HEK293 and HepG2 cells under hyperosmotic stress / Chapter 1.1 --- Introduction --- p.22 / Chapter 1.1.1 --- Cellular response to hyperosmotic stress --- p.22 / Chapter 1.1.2 --- Methods to study the responses of cells under hyperosmotic stress --- p.24 / Chapter 1.2 --- Materials --- p.26 / Chapter 1.2.1 --- Cell culture media --- p.26 / Chapter 1.2.2 --- Buffers for RNA use --- p.26 / Chapter 1.2.3 --- Buffers for DNA use --- p.27 / Chapter 1.2.4 --- Other chemicals --- p.27 / Chapter 1.3 --- Methods --- p.28 / Chapter 1.3.1 --- Culture of HEK293 and HepG2 cells --- p.28 / Chapter 1.3.2 --- Hyperosmotic stress on HEK293 and HepG2 cells --- p.29 / Chapter 1.3.3 --- MTT assay --- p.29 / Chapter 1.3.4 --- Total RNA extraction --- p.30 / Chapter 1.3.5 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 1.3.6 --- Polymerase chain reaction (PCR) --- p.31 / Chapter 1.3.7 --- Quantification of PCR products --- p.31 / Chapter 1.3.8 --- Statistical analysis --- p.33 / Chapter 1.4 --- Results --- p.34 / Chapter 1.4.1 --- Viability of HEK293 and HepG2 cells under hyperosmotic stress --- p.34 / Chapter 1.4.2 --- Validation of RNA quality --- p.34 / Chapter 1.4.3 --- Validation and determination of PCR conditions --- p.40 / Chapter 1.4.4 --- Inducibility of antiquitin in HEK293 cells under hyperosmotic stress / Chapter 1.4.5 --- Inducibility of antiquitin in HepG2 cells under hyperosmotic stress --- p.43 / Chapter 1.4.6 --- Inducibility of aldose reductase under hyperosmotic stress --- p.43 / Chapter Chapter 2 --- "In silico studies of human antiquitin promoter, genomics sequences and open reading frame" --- p.54 / Chapter 2.1 --- Introduction --- p.54 / Chapter 2.1.1 --- Eukaryotic promoters --- p.55 / Chapter 2.1.2 --- Key events in transcriptional initiation --- p.55 / Chapter 2.1.3 --- Alternative splicing of mRNA --- p.57 / Chapter 2.1.4 --- Bipartite nuclear localization signal (NLS) --- p.57 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Putative promoter studies of human antiquitin --- p.60 / Chapter 2.2.2 --- Putative promoter studies of Arabidopsis thaliana antiquitin --- p.60 / Chapter 2.2.3 --- Analysis for the alternative splicing of human antiquitin mRNA --- p.60 / Chapter 2.2.4 --- Analysis for the nuclear localization signal (NLS) of human antiquitin amino acid sequence --- p.61 / Chapter 2.2.5 --- Nucleotide / amino acid sequence analyses --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- Computer search for the putative cis-acting elements on human antiquitin promoter --- p.62 / Chapter 2.3.2 --- Comparison of cis-acting elements found on human antiquitin promoter with those on Arabidopsis thaliana antiquitin promoter --- p.62 / Chapter 2.3.3 --- Possibilities of alternative splicing isoforms of human antiquitin / Chapter 2.3.4 --- Possibilities of bipartite nuclear localization signals on human antiquitin protein --- p.83 / Chapter Chapter 3 --- Overexpression of antiquitin in HEK293 and HepG2 cells and their characterization / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- Cell cycle of a human somatic cell --- p.88 / Chapter 3.1.2 --- Detection of changes in the transcriptome --- p.90 / Chapter 3.1.3 --- Human genome U133 Plus 2.0 array --- p.95 / Chapter 3.1.4 --- Detection of changes in the proteome --- p.96 / Chapter 3.1.5 --- MALDI-TOF MS --- p.97 / Chapter 3.2 --- Materials --- p.99 / Chapter 3.2.1 --- Solutions for cell culture use --- p.99 / Chapter 3.2.2 --- Solutions for cloning --- p.99 / Chapter 3.2.3 --- Buffers for cell cycle analysis --- p.99 / Chapter 3.2.4 --- Buffers for two-dimensional (2D) electrophoresis --- p.100 / Chapter 3.2.5 --- Solutions for silver staining --- p.101 / Chapter 3.2.6 --- Solutions for Coomassie blue protein staining --- p.102 / Chapter 3.2.7 --- Solutions for Western blotting --- p.102 / Chapter 3.2.8 --- Solutions for mass spectrometry --- p.103 / Chapter 3.3 --- Methods --- p.104 / Chapter 3.3.1 --- Hypoosmotic stress --- p.104 / Chapter 3.3.2 --- Heat shock --- p.104 / Chapter 3.3.3 --- Oxidative stress treatment / Chapter 3.3.4 --- Chemical hypoxia --- p.104 / Chapter 3.3.5 --- Treatment of forskolin --- p.106 / Chapter 3.3.6 --- Culture of SHSY5Y cells and its differentiation --- p.106 / Chapter 3.3.7 --- Cloning of pBUDCE4.1/ATQ --- p.106 / Chapter 3.3.8 --- PCR product purification --- p.107 / Chapter 3.3.9 --- Preparation of pEGFP.N1 vector for co-transfection --- p.109 / Chapter 3.3.10 --- Transfection of HEK293 and HepG2 cells --- p.109 / Chapter 3.3.11 --- Assays to characterize transient transfected HEK293 and HepG2 cells --- p.110 / Chapter 3.3.11.1 --- Transfection efficiency monitoring --- p.110 / Chapter 3.3.11.2 --- Cell cycle analysis --- p.112 / Chapter 3.3.11.3 --- Cell doubling time measurement --- p.112 / Chapter 3.3.11.4 --- Stress responsiveness --- p.113 / Chapter 3.3.11.5 --- Oligonucleotide array analysis --- p.113 / Chapter 3.3.11.5.1 --- Total RNA extraction --- p.113 / Chapter 3.3.11.5.2 --- Oligonucleotide array preparations --- p.113 / Chapter 3.3.11.5.3 --- Data analysis --- p.114 / Chapter 3.3.11.6 --- Two-dimensional (2D) electrophoresis --- p.115 / Chapter 3.3.11.6.1 --- Total protein extraction --- p.115 / Chapter 3.3.11.6.2 --- Protein quantification --- p.115 / Chapter 3.3.11.6.3 --- First dimension electrophoresis: isoelectric focusing (IEF) --- p.115 / Chapter 3.3.11.6.4 --- Second dimension electrophoresis: SDS- --- p.116 / Chapter 3.3.11.6.5 --- Silver staining --- p.116 / Chapter 3.3.11.6.6 --- Spots detection --- p.117 / Chapter 3.3.11.7 --- Preparations of samples for MALDI-TOF MS --- p.117 / Chapter 3.3.11.7.1 --- Silver de-staining --- p.117 / Chapter 3.3.11.7.2 --- In-gel tryptic digestion --- p.118 / Chapter 3.3.11.7.3 --- Peptide extraction --- p.118 / Chapter 3.3.11.7.4 --- ZipTip® samples desalting and concentrating --- p.119 / Chapter 3.3.11.7.5 --- MALDI-TOF MS --- p.119 / Chapter 3.3.11.8 --- Western blotting --- p.119 / Chapter 3.3.11.8.1 --- Antibodies probing --- p.120 / Chapter 3.3.11.8.2 --- Enhanced chemiluminescence's (ECL) assay --- p.121 / Chapter 3.4 --- Results --- p.122 / Chapter 3.4.1 --- Inducibility of antiquitin in HEK293 cells under xenobiotic stimulus --- p.122 / Chapter 3.4.2 --- Inducibility of antiquitin in HEK293 and HepG2 cells under chemical hypoxia --- p.122 / Chapter 3.4.3 --- Inducibility of antiquitin in HEK293 and HepG2 cells under hypoosmotic stress --- p.122 / Chapter 3.4.4 --- Inducibility of antiquitin in HEK293 and HepG2 cells under heat shock --- p.122 / Chapter 3.4.5 --- Inducibility of antiquitin in HEK293 and HepG2 cells under forskolin challenge --- p.128 / Chapter 3.4.6 --- Expression of antiquitin in differentiating SHSY5Y cells by retinoic acid and N2 supplement --- p.128 / Chapter 3.4.7 --- Overexpression of antiquitin in HEK293 and HepG2 cells --- p.128 / Chapter 3.4.8 --- Viability of transfected HEK293 and HepG2 cells under hyperosmotic stress --- p.136 / Chapter 3.4.9 --- Cell doubling times of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.10 --- Cell cycle analysis of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.11 --- "Western blot analysis of cyclin D, cyclin A and cyclin B of transfected HEK293 and HepG2 cells" --- p.148 / Chapter 3.4.12 --- RNA quality control tests for oligonucleotide array analysis --- p.148 / Chapter 3.4.13 --- Oligonucleotide array analysis on transfected HEK293 and HepG2 cells --- p.155 / Chapter 3.4.14 --- Two-dimensional electrophoresis of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.15 --- MALDI-TOF MS of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.16 --- Genes and proteins upregulnted in the antiquitin transfected HEK293 and HepG2 cells --- p.190 / Discussion --- p.197 / Reference --- p.221 / Appendix Materials used in the project --- p.243

by Tang Wai Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 91-110). / Abstracts in English and Chinese. / Acknowledgements / 論文摘要 / Abstract / Abbreviations / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Aldehyde Dehydrogenase Extended Family --- p.1 / Chapter 1.1.1 --- Phylogenetic Tree --- p.2 / Chapter 1.1.2 --- Physiological Functions --- p.4 / Chapter 1.1.3 --- Structural Conservations --- p.7 / Chapter 1.2 --- ALDH-1 and ALDH-2 --- p.9 / Chapter 1.3 --- Antiquitin --- p.11 / Chapter 1.4 --- Osmoregulation --- p.14 / Chapter 1.4.1 --- Osmoprotectant --- p.14 / Chapter 1.4.2 --- Betaine Aldehyde Dehydrogenase --- p.15 / Chapter 1.5 --- Objectives of the Present Study --- p.18 / Chapter Chapter 2 --- Purification and Characterization of Seabream ALDH-2 and Antiquitin --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials --- p.21 / Chapter 2.3 --- Methodology / Chapter 2.3.1 --- Preparation of Crude Tissue Extract --- p.22 / Chapter 2.3.2 --- Synthesis of α-Cyanocinnamate Sepharose --- p.22 / Chapter 2.3.3 --- Synthesis of p-Hydroxyacetophenone Sepharose --- p.23 / Chapter 2.3.4 --- Purification of ALDH-2 --- p.23 / Chapter 2.3.5 --- Purification of Antiquitin --- p.24 / Chapter 2.3.6 --- Enzyme and Protein Assays --- p.24 / Chapter 2.3.7 --- Electrophoretic Procedures / Chapter 2.3.7.1 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.26 / Chapter 2.3.7.2 --- Native PAGE --- p.27 / Chapter 2.3.7.3 --- Isoelectric focusing (IEF) --- p.27 / Chapter 2.3.8 --- N-terminal Amino Acid Sequencing --- p.28 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Tissue Distribution of ALDH --- p.29 / Chapter 2.4.2 --- Purification and Molecular Properties of ALDH-2 --- p.31 / Chapter 2.4.3 --- Kinetic Properties of ALDH-2 --- p.42 / Chapter 2.4.4 --- Purification and Molecular Properties of Antiquitin --- p.49 / Chapter 2.4.5 --- Kinetic Properties of Antiquitin --- p.54 / Chapter Chapter 3 --- Discussion / Chapter 3.1 --- Tissue Distribution --- p.66 / Chapter 3.2 --- N-terminal Amino Acid Sequencing --- p.67 / Chapter 3.3 --- Purification of Seabream ALDH --- p.68 / Chapter 3.3.1 --- Separation of Two ALDH isoforms --- p.69 / Chapter 3.3.2 --- Binding Affinity of α-Cyanocinnamate Sepharose --- p.70 / Chapter 3.3.3 --- Purification --- p.72 / Chapter 3.4 --- Electrophoretic Properties --- p.73 / Chapter 3.5 --- pH and Temperature Stability --- p.74 / Chapter 3.6 --- Substrate Specificity --- p.77 / Chapter 3.7 --- Possible Functions of Antiquitin --- p.80 / Chapter 3.8 --- Future Prospects --- p.84 / Chapter Chapter 4 --- Conclusion --- p.90 / Chapter Chapter 5 --- References --- p.91